Under support of this grant, our laboratory has investigated 2 bicoid-related homeodomain transcription factor, referred to as Pitx1 and Pitx2 that have proved to be critical regulators of development of the cardiac outflow tract and heart, identifying a novel Wnt/Dvl/beta-catenin-Pitx2 pathway that is required for correct development of the cardiac neural crest. Our laboratory is now focused on dissection of transcriptional events that regulate specific aspects of development of the cardiovascular system and understanding the underlying molecular mechanisms and gene ontology programs that are required for these events. Our investigation of transcriptional repression and corepressors has led to the discovery of an unexpected role of the nuclear receptor corepressor, SMRT, in heart development and the identification of an unexpected mechanism for activation by regulated DNA binding transcription factors. In this competitive renewal, we will develop new strategies for genome-wide localization of regulatory transcription factors and cofactors that, in coupled with siRNA/RNA profiling strategies, will permit elucidation of direct target genes for specific transcriptional factors and signaling pathways important in cardiovascular development. Using these technologies, we will define the gene ontology programs by which Pitx2 and SMRT regulate cardiovascular development. The discovery in cardiovascular and macrophage developmental programs of a novel strategy to enforce repression checkpoints that are critical in regulation of both macrophage, epicardium and myocardium, will be pursued to define the underlying molecular mechanisms of cofactor exchange. These studies should add to our knowledge of the molecular basis of early events critical for heart and outflow tract development, and should provide a basis for evaluating new approaches for modulating these events. These studies are likely to have direct implications for several human cardiovascular defects and diseases.